Expression and purification of a recombinant adenovirus fiber knob in a baculovirus system

Autor(es)

Farinha-Arcieri, Luis E. ^[1] ; Porchia, Bruna M. ^[1] ; Carromeu, Cassiano ^[1] ; Simabuco, Fernando M. ^[1] ; Tamura, Rodrigo E. ^[1] ; Ferreira, Luis C. S. ^[1] ; Zerbini, Luiz F. ^[2] ; Ventura, Armando M. ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Inst Ciencias Biomed, Dept Microbiol, BR-05508900 Sao Paulo - Brazil

^[2] Harvard Inst Med, BIDMC, Boston, MA - USA

• Tipo de documento: Artigo Científico
• Fonte: Intervirology; v. 51, n. 3, p. 189-195, 2008.
• Citações Web of Science: 3

Resumo

Objectives: To construct a recombinant baculovirus expressing the fiber knob domain of human adenovirus type 2 modified by the insertion of a foreign peptide, purify this protein after its production in insect cells, and to test its properties. Methods: Recombinant baculoviruses expressing the fiber knob were produced in Sf9 cells. The recombinant fiber knob was recovered from culture supernatants of infected cells and purified by a combination of Ni-NTA and ion-exchange chromatography. Results: Fiber knob was recovered from the culture media as a soluble protein. In the system used, the fiber knob is expressed fused with the V5 epitope and a histidine tag, which allowed purification by Ni-NTA chromatography. The protein was further purified by ion-exchange chromatography. We show that the recombinant fiber knob produced, with 31 extra amino acids in the C-terminus, can oligomerize and bind to the adenovirus receptor CAR, as it can block the infection of a recombinant type 5 adenovirus. Conclusions: The modified form of the fiber knob, produced in insect cells and purified by Ni-NTA and ion-exchange chromatography, retains the properties of oligomerization and binding to the fiber natural receptor, CAR. This construct has the potential to be a new adjuvant. Copyright (C) 2008 S. Karger AG, Basel. (AU)

• Processo FAPESP: 03/02041-4 - Expressao de proteinas e peptideos de rsv e hiv para estudos moleculares e de imunizacao.
• Beneficiário: Armando Morais Ventura
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 06/59976-3 - Expressao de peptideos de hrsv e hiv para estudos de imunizacao e moleculares.
• Beneficiário: Armando Morais Ventura
• Modalidade de apoio: Auxílio à Pesquisa - Regular